Spontaneous mutations of the human ATP-binding cassette breast cancer resistance protein, BCRP (ABCG2), convey tumor cell resistance by exporting classical anti-cancer agents, often resulting in a fatal relapse of chemotherapy. Human ABCG2 is a homodimeric integral membrane protein with a unique and compact "half-transporter" design and does not require any glycosylation to function as a drug export pump. These characteristics may make this protein the best candidate among human multidrug export pumps for high-resolution structure determination. The protocols for the expression of low milligram quantities of ABCG2 from the yeast Pichia pastoris have already been developed. Production of even higher quantities of protein from cell-free systems will be optimized now that non-ionic detergents that stabilize hABCG2 have already been identified. The goal of this proposal is to solve the structure of human ABCG2 by x-ray crystallography to visualize the mechanisms by which this clinically important protein drives anti-cancer pharmaceuticals out of cells. "Click" chemistry techniques will be applied to purified ABCG2 protein and protein crystals to identify building blocks of specific inhibitors to this drug export pump. A high-resolution crystal structure will be used in combination with the output from the "click" chemistry to enable the design of next-generation anti-cancer pharmaceuticals that will specifically block ABCG2 activity and increase the effectiveness of chemotherapy in several human cancers. [unreadable] [unreadable] [unreadable]